**3. Microbes – The enemy?**

126 New Approaches to the Study of Marine Mammals

A microbe that manages to penetrate the subcutaneous layers of the skin will be met by the second line of defence; the innate or non-specific immune system. This defence mechanism involves the production pro-inflammatory substances, such as chemokines by the lipokeratinocytes, resulting in the migration of immune cells such as leucocytes to the site of infection. Leucocytes include among others, the phagocytes (macrophages, neutrophils and dendritic cells). Upon detection of the foreign proteins originating from potential bacterial, fungal or parasitic invaders, Langerhans cells will phagocytise these antigens and migrate to adjacent lymph nodes. In the lymph nodes, these cells will develop into mature dendritic cells which will process the antigen (into smaller fragments), to activate the adaptive immune system (lymphocytes) [14]. On top of this chemical defence mechanism, nonspecific antimicrobial substances such as lysozyme and the peptide β-defensin were found in cetacean integument [15]. It was found that lysozyme occurs between the layers of the stratum corneum, within cells of the stratum spinosum, dermis and endothelial cells of the dermal blood vessels. Also, β-defensin was found to be concentrated in the upper five or six layers of the stratum corneum, as well as within the cells of the upper stratum spinosum.

In addition to the above-mentioned non-specific defence mechanisms, intra-epidermal lymphocytes indicate the presence of the adaptive immune system in the epidermis of mammals. These specialized T cells have powerful cytolytic and immuno-regulatory effects on antigens and will confine antigens that overcame the first and second lines of skin defence [14]. Cells associated with this specific or adaptive immune response were also detected in cetacean skin studies [13]. Zabke and Romano [13] reported that their study on dolphin skin revealed the presence of MHC II (+) antigen cells, predominantly situated in the dermal papillae, along the epidermal-dermal border. These cells were found to have a dendritic-like morphology and form patterns, similar to those of Langerhans cells. The latter are known primary antigen presenting cells in the integument of terrestrial mammals, and thus the authors concluded that they were most likely Langerhans cells and not macrophages or dendritic cells. The latter two types were found deeper into the dermis. Zabke and Romano [13] further suggested that pathogen invasion resulting from a wound may lead to an inflammatory response, causing immune cells (neutrophils, macrophages and/or lymphocytes) to migrate from the dermal papillae to the site of infection. However, the authors indicated that inflammation is usually absent in these animals, because this barrier is normally sufficient against small injuries sustained via interactions with other dolphins. The authors further noted that wound healing in dolphins is not accompanied by scab formation. In these mammals hydrophobic changes within in the stratum spinosum, causes rapid sloughing and

replacement with cells of the stratum germinativum undergoing mitosis.

strategies and unique properties in the prospective invader.

From the above it is evident that cetacean skin is an effective physicochemical barrier. To overcome such a challenging obstacle would require a failure of the barrier itself, or creative

*Immune response to cutaneous fungal infections.* The type of fungal invader, whether a unicellular yeast or hyphal fungus, as well as anatomical site of infection, will determine the immune response of the host [16,17]. Yeast cells are usually phagocytosed, whereas the A limited number of organisms have the ability to degrade and utilize keratin, the key structural component of mammalian skin [18]. These include a few insect species, as well as a number of bacteria and fungi. Higher vertebrates are also not known to digest keratin [11]. Microbes degrade keratin by the secretion of extracellular proteolytic enzymes, known as keratinases; members of the serine proteinase group of enzymes [19, 20]. These enzymes are robust with a wide temperature and pH activity range, and have the ability to hydrolyze both natural and denatured keratin [21]. Keratinases from the fungus *Microsporum gypseum* were found to cleave the disulfide bridges in the keratin (sulfitolysis), which were followed by a further attack on the keratin structure by extracellular proteases [18]. Tsuboi and coworkers [22] found a keratinolytic proteinase in another fungus, i.e. *Trichophyton mentagrophytes,* with an optimal pH of 4.5 for keratin and 3.9 for haemoglobin. They showed that this fungus could potentially invade healthy skin (with a weakly acidic pH), by breaking down the keratin and thereby making it possible for the organism to invade the stratum corneum. This ability to degrade keratin, and other molecules associated with skin, can be regarded as a putative microbial virulence factor [20].

Keratinophilic (keratin loving) fungi (Figure 2) represent the largest group of organisms with the ability to degrade and utilize keratin as a source of carbon and nitrogen [11, 16]. These fungi commonly occur in soil and sewage sludge, which contain high concentrations of keratin remnants with specific physiochemical properties and associated microbial populations [23]. Releasing sewage sludge into the environment, or using it for fertilizing purposes, can therefore lead to spreading of potentially pathogenic fungi into new

ecosystems. Plants, humans and animals are subsequently exposed to a variety of potentially infectious microbes. Fungal genera known to harbour keratinophilic species include: *Acremonium, Alternaria. Aspergillus, Candida, Chaetomium, Chrysosporium, Cladosporium, Curvularia, Fusarium, Geotrichum, Gliocladium, Gymnoascus, Microsporum, Monoascus, Mucor, Paecilomyces, Penicillium, Scopulariopsis, Sporothrix, Trichoderma, Trichophyton,* as well as *Verticillium* [16, 23-25].

Cutaneous Lesions in Cetaceans: An Indicator of Ecosystem Status? 129

this regard for commercial purposes, specifically for the biodegradation of feathers in the poultry industry [21, 26]. Other bacterial genera with keratinolytic ability include: *Lysobacter, Nesternokia, Kocurica, Microbacterium, Vibrio, Xanthomonas, Stenotrophomonas* and

Skin lesions in cetaceans have been reported since the 1950's [2]. Some examples of the

Skin lesions containing *Staphylococcus* were reported for the first time in cetaceans in 1988 [27]. In that study, two isolates of *Staphylococcus* were obtained from purulent tissue that occurred in two captive dolphins with multiple, suppurating lesions, and subsequently

A study conducted over a four year period found that a range of microbes were associated with lesional and non-lesional skin in a group of bowhead whales (*Balaena mysticetus*), characterized by dozens to hundreds of roughened areas on their skin surfaces [28]. The majority of microbes, isolated during the study, were associated with the lesional skin: 56% of Gram positive bacteria, 75% of Gram negative bacteria and 64% of the yeasts. Also, the lesional skin was characterized by the presence of *Corynebacterium* spp.*, Acinetobacter* spp., as well as representatives of *Moraxella*. *Candida* spp. were the dominant yeast species, followed by representatives of *Cryptococcus* and *Rhodotorula.* Subsequent tests on the isolates showed the production of enzymes able to cause necrosis by microbes originating from both lesional and non-lesional skin. These whales occurred in regions with increased industrial activities (gas and oil exploration) in the Beaufort Sea; the authors speculated that the roughened skin

A study examining skin diseases among wild cetaceans from British waters found 69% of individuals to be affected [29]. The authors reported wounds and other traumatic injuries, as well as lesions caused by pox and herpes viruses, as well as bacteria, ectoparasites and nonspecific ulcers. Concerns that the lesions were associated with pollution were raised but not

Henk and Mullan [30] examined 23 bowhead whales and reported shallow lacerations, circular depressions and epidermal sloughing on these whales' skin. The authors also found abundant bacteria and diatoms associated with these lesions, and even higher numbers where the stratum spinosum was exposed. The bacterial isolates were found to include cocci, bacilli and filamentous spp., with increasing numbers associated with higher levels of necrotic decay. Protozoa and fungi were also observed and also increased in incidence with more disturbed epidermal surfaces. The authors also identified several erosive enzymes from these microbes and suggested an association between the whales' skin and spilled oil. A pygmy sperm whale (*Kogia breviceps*) and an Atlantic white-sided dolphin (*Lagenorhynchus acutus*) were reported with mycotic dermatitis, in the form of raised, firm, erythemous, cutaneous nodules on parts of their bodies [31]. The dermatitis cases were the result of

**4. Case studies of skin lesions in cetaceans – The signs?** 

areas might have been associated with the adherence of spilled oil [28].

steady stream of reports in later years are presented below.

described as a new species, *Staphylococcus delphini*.

*Chryseobacterium* [24].

confirmed [29].

**Figure 2.** Light micrographs of keratinophilic fungi. a. Fruit body of *Chaetomium murorum* with long ascomatal setae; b. ascomatal setae and ascospores of *Chaetomium murorum*; c. conidia of *Alternaria alternata*; d. Fertile hyphae of *Chrysosporium keratinophilum* bearing conidia

Some keratinophilic fungi (Figure 2) are known to be pathogenic, and often the cause of cutaneous skin infections. These include the so-called dermatophytes, belonging to the genera *Epidermophyton, Microsporum* and *Trichophyton,* as well as non-dermatophytes such as *Aspergillus, Candida, Fusarium* and *Scopulariopsis* spp*.* Moreover, De Hoog et al. [16] remarked that more cases of fungal infections seem to be caused by fungal spp. formerly known as being saprobic, and appear to be associated with the increasing numbers of immuno-compromised patients. These fungi are mostly opportunists that cause infection when the immune system of the host is breached [16, 17]. Interestingly, of more than 100 000 known fungal species, only about 100 have been reported regularly, as infectious agents of animals, as well as humans [16].

Certain species of the bacterial genus *Bacillus* also have the ability to produce keratinases. *Bacillus cereus, Bacillus licheniformis* and *Bacillus subtilis* have been identified and studied in this regard for commercial purposes, specifically for the biodegradation of feathers in the poultry industry [21, 26]. Other bacterial genera with keratinolytic ability include: *Lysobacter, Nesternokia, Kocurica, Microbacterium, Vibrio, Xanthomonas, Stenotrophomonas* and *Chryseobacterium* [24].
